Reversible protein phosphorylation is a major regulatory mechanism in a cell. A chemical-genetic strategy to conditionally inactivate protein kinases has been developed recently. Mutating a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors. The inhibitor can only bind to the mutant kinase and not to any other wild-type kinase, allowing specific inactivation of the modified kinase. Here, we describe a protocol to construct conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe. This protocol can be completed in about 3 weeks and should be applicable to other organisms as well.